Barley which is one example of plants is an essential agricultural crop for feed and for producing food and drink (beer, whisky, etc.), and is worldwide cultivated and consumed. In accordance with many uses for barley, various breeding in barley have heretofore been made. One conventional breeding in barley comprises selecting some effective varieties from artificial or natural mutants followed by combining them through mating or the like to thereby find out from a number of the resulting progeny the hybrids capable of expressing the intended phenotype. However, as comprising mating, the breeding of this type is problematic in that the genotype to be introduced is limited to one for relative mating and that it takes a long period of time to obtain the intended hybrids.
On the other hand, with the recent development in biotechnology such as genetic engineering technology and cell technology, a system for directly introducing a desirable gene into plants is being established even for barley, and is expected to be one capable of overcoming the problems in the conventional breeding (for example, BIOTECHNOLOGY 13, 248, 1995 is referred to for barley for brewing). The system requires a tissue-specific promoter. In this, precisely, where a foreign gene is introduced into a plant, the gene is required to be sufficiently expressed in the intended tissue in good time. For this purpose, a tissue-specific promoter must be linked to the foreign gene to thereby make the gene expressible under the control of the promoter.
The present invention is to provide a promoter capable of being specifically expressed in plant seeds. As one example, we, the inventors of the present invention have succeeded in the isolation of a promoter region that acts to control the transcription of a barley .beta.-amylase gene and also in the analysis of the nucleotide sequence of the promoter region.
Barley .beta.-amylase is a .beta.-amylase obtainable from barley seeds (1,4-.alpha.-D-glucanmaltohydrolase EC 3.2.1.2!), which is known as an enzyme usable, like soybean .beta.-amylase, in the industrial production of maltose for injection and maltose for food and drink. It is also known that barley may be germinated to give malt which may be a raw material for beer and liquors. .beta.-amylase existing in malt is one of the most important enzymes for the saccharification of starch in the step of mashing.
Regarding the gene of barley .beta.-amylase, the complete sequence of cDNA of a variety of barley, Hiproly, which comprises 1754 bases, has been reported, and the amino acid sequence thereof comprising 535 residues has also been deduced (see Eur. J. Biochem., 169, 517, 1987).
In addition, the complete sequence of cDNA of a variety of barley, Haruna Nijo, which comprises 1775 bases, has been reported, and the amino acid sequence thereof comprising 535 residues has also been deduced (see J. Biochem., 115, 47, 1994; Japanese Patent Application Laid-Open No. 6-303983). Further, the complete sequence of the structural gene region of the chromosome DNA of the variety, Haruna Nijo, which comprises 3825 bases, has been reported (see Japanese Patent Application No. 7-92004).
However, regarding the promoter region that acts to control the transcription of such a .beta.-amylase gene, there is no report referring to the isolation of the promoter gene, let alone the analysis of the nucleotide sequence thereof.
We, the present inventors, having attempted to isolate the promoter region of a barley .beta.-amylase gene that may be actively expressed in barley developing seeds, thereby utilizing it in the improvement of barley seeds or in the production of products in barley seeds, have earnestly studied to attain this object, and, as a result, have completed the present invention.
According to the present invention, a desired foreign gene and a terminator therefor can be linked to the downstream site of the promoter region obtained, and introduced into plant such as barley, whereby the foreign gene can be expressed in the plant developing seeds. Thus, the promoter region can be utilized in the improvement of seeds of barley and other plants and also in the production of substances in such seeds.